Many potent antagonists of LHRH (the luteinizing hormone releasing hormone, pGlu,His,Trp,Ser,Tyr,Gly,Leu,Arg,Pro,Gly-NH.sub.2) have structural features which cause release of histamine from mast cells [Karten et al., 1986]. These features include a group of hydrophobic amino acids at the N-terminal and strongly basic residues in positions 6 and 8, notably D-Arg.sup.6, Arg.sup.8. A prime example of this class of antagonists is [N-Ac-D-2-Nal.sup.1,D-4F-Phe.sup.2 D-Trp.sup.3,D-Arg.sup.6 ]-LHRH [Schmidt et al.]. Some antagonists bind to rat peritoneal mast cells and membrane preparations and that the binding was related to the release of histamine [Sundaram et al.].
______________________________________ Abbreviations for the unnatural amino acids mentioned herein ______________________________________ Abu = 2-aminobutyric acid (AcDSer)Lys = N.sup..epsilon. -(N-acetyl-D-seryl)lysine (DSer)Lys = N.sup..epsilon. -(D-seryl)lysine 2-Nal = 3-(2-naphthyl)alanine 3-Pal = 3-(3-pyridyl)alanine 3-Qal = 3-(3-quinolyl)alanine AOPP = 2-(31-amino-2'-oxo-1'-pyrrolidino)-4- methylpentanoic acid Aze = azetidine-2-carboxylic acid Cit = citrulline Cl.sub.2 Phe = 3-(3,4-dichlorophenyl)alanine cPzACAla = cis-3-(4-pyrazinylcarbonylaminocyclohexyl)alanine ILys = N.sup..epsilon. -isopropyllysine NicLys = N.sup..epsilon. -nicotinoyllysine p-FPhe = 3-(4-fluorophenyl)alanine pClPhe = 3-(4-chlorophenyl)alanine PicLys = N.sup..epsilon. -picolinoyllysine Ptf = 3-(4-trifluoromethylphenyl)alanine PzAla = 3-pyrazinylalanine PzAPhe = 3-(4-pyrazinylcarbonylaminophenyl)alanine PzcLys = N.sup..epsilon. -pyrazinylcarbonyllysine PzLys = N.sup..epsilon. -pyrazinylcarbonyllysine ______________________________________
The present inventors have recently developed Antide (analog 1, Table III) which lacked strongly basic residues and which showed high potency and negligible histamine release [Ljungqvist et al, 1987]. Further evaluation of the anaphylactoid activity of Antide showed that Antide "represents a new generation of LHRH antagonists with an improved safety margin" [Phillips et al.].
Prolonged duration of inhibition of gonadotropin secretion in overectomized monkeys using single [Leal et al.1988] or multiple [Leal et al, 1989] doses of Antide has also been established.
The same group observed a long-term suppression of testosterone secretion in male monkeys after a single dose of Antide [Edelstein et al.].
The mechanism(s) for this long-term action of Antide seems to include binding to serum proteins for a peripheral depot effect, and structural stability to enzymic cleavage. Binding to proteins was shown by studies using a radioreceptor assay [Danforth et al.].
If a depot effect, regardless of mechanism, is at least partially responsible for the long duration of action of Antide, the relatively poor water solubility at physiological pH may actually be an advantage. There are, however, reports that low water solubility has caused problems [Lee et al., Miller et al.].
It was thus considered of importance to modify Antide and some potent analogs [Ljungqvist et al., 1987; Ljungqvist et al., 1988] in order to increase their water solubility and to study how this would effect the biological activities of the resulting analogs.
Lunenfield et al. summarized four different rationales for potential therapy of clinical modalities. They are: (1) to suppress steroid-dependent mechanisms of malignancies and endometriosis; (2) to inhibit precocious puberty, etc.; (3) to control gonadotropin secretion in ovulation, etc.; (4) to exploit other effects depending upon future proof of applicability. The existing wide-scale clinical use of LHRH agonists is a background for potential uses of antagonists, but the lower activities of antagonists, by one-thousandth that of agonists means that per unit dose the potency of the presently known antagonists need to be increased up to ten-fold for promising clinical use.
In the early years, antagonists such as [D-Phe.sup.2, Pro.sup.3, DTrp.sup.6 ] LHRH (Humphries et al., 1976) showed AOA.sub.100 at 750 .mu.g/rat. Introduction of basic D-amino acids in position 6 resulted in a significant increase of antiovulatory activity. Antagonists like [N-Ac-D-2-Nal.sup.1 ; D-pClPhe.sup.2, D-Trp.sup.3, D-Arg.sup.6, D-Ala.sup.10 ] LHRH by Horvath et al and [N-Ac-D-2-Nal.sup.1, D-pClPhe.sup.2, D-3-Pal.sup.3, D-Arg.sup.6, Trp.sup.7, D-Ala.sup.10 ] LHRH by Folkers et al. (1984) were examples of antagonists with AOA (antiovulatory activity) of 100% at ca. 0.5 .mu.g/rat.
However, the most potent D-Arg.sup.6 -containing antagonists produced edema in the face and extremities [Schmidt et al.; Morgan et al.] and a dose-related wheal response [Hahn et al.]. These undesired effects apparently caused by release of histamine from mast cells [Hook et al.] have been ascribed to the presence of strongly basic residues in position 6 and 8, e.g., D-Arg.sup.6, Arg.sup.8, and a cluster of hydrophobic amino acids at the N-terminal [Nikola et al., Roeske et al.].
With new emphasis on decreasing the histamine releasing activity, structural modification of antagonists was focused primarily on reducing basicity in positions 6 and 8. This goal was achieved by different approaches. Folkers et al. 1986, introduced D-3-Pal.sup.6 instead of D-Arg.sup.6 and obtained the relatively potent antagonist [N-Ac-D-2-Nal.sup.1, D-pClPhe.sup.2, D-3-Pal.sup.3, Arg.sup.5, D-3-Pal.sup.6, D-Ala.sup.10 ] LHRH, but the histamine release was undesirable. Suppression of histamine release occurred by introduction of D-ureidoalkyl amino acids such as D-citrulline or D-homocitrulline at position 6 by Bajusz et al., but the most active antagonist of their series [N-Ac-D-2-Nal.sup.1, DpClPhe.sup.2, D-Trp.sup.3, D-Cit.sup.6, D-Ala.sup.10 ] LHRH, caused 100% AOA in doses as high as 3 .mu.g/rat.
Combining aspects of safety and AOA was achieved by Ljungqvist et al., 1987, with a new class of antagonists which featured acylated Lys residues in positions 5 and 6 in combination with alkylated Lys in position 8. The prominent example of this class of antagonists is Antide, [N-Ac-D-2-Nal.sup.1, D-pClPhe.sup.2, D-3-Pal.sup.3, NicLys.sup.5, D-NicLys.sup.6, Ilys.sup.8, D-Ala.sup.10 ] LHRH which completely inhibited ovulation at 1 .mu.g/rat and had an ED.sub.50 for histamine release 300 .mu.g/ml. Antagonists with AOA superior to Antide with acylated aminocyclohexylalanines and acylated lysines in position 5 and 6 were next reported; Ljungqvist et al., 1990.
The synthesis and bioassays of some new analogs with improved water solubility and biological effectiveness are described herein.
Antagonists of the hypothalamic luteinizing hormone releasing hormone (LHRH), decapeptide, pClu,His,Trp,Ser,Tyr,Gly,Leu,Arg,Pro,GlyNH.sub.2, have been proposed for use in the control of fertility and in the treatment of hormone dependent tumors. A few thousand analogs of LHRH have been reported since its structure was elucidated in 1971 [Amoss et al,. Schally et al.]. The first thirteen years witnessed the development of the Nal-Arg generation of antagonists [Karten et al., 1986], but they were also potent, in vitro, to release histamine [Schmidt et al.]. With that knowledge, emphasis was then placed on reducing the histamine releasing potency while maintaining or preferably increasing gonadotropin suppressive potency.